One of the most critical activities of the ribosome is maintenance of the translational reading frame. Although recent structures have revealed numerous interactions between the ribosome and its primary substrates, tRNA and mRNA, how these interactions contribute to reading frame maintenance remains unclear. Understanding this ribosomal activity will directly impact research on programmed ribosomal frameshifting, a class of genetic regulatory mechanisms employed by numerous organisms and viruses, including medically relevant retroviruses and coronaviruses. This proposal has three specific aims: (1) Identify interactions between ribosomal RNA (rRNA) and tRNA important for accurately coupled movement of tRNA and mRNA during translocation. (2) Identify determinants of translocation catalyzed by elongation factor G and by the antibiotic sparsomycin. (3) Use mutant tRNA molecules with expanded anticodons to investigate the contribution of codon-anticodon interactions to reading frame recognition and maintenance. These studies will provide novel insights into the molecular mechanism of reading frame maintenance by the ribosome, a fundamental aspect of gene regulation. Furthermore, this research may significantly contribute to the design of drugs that target HIV.